Migration of Melanoma Cells through an in vitro model of the Blood-Brain Barrier Endothelium

Abstract

Background and aims: Melanoma is an aggressive skin cancer with high propensity to metastasise to the brain. However, the brain is protected from harmful substances in the blood, due to the presence of the highly specialised blood-brain barrier (BBB). Brain endothelial cells are the primary component of the BBB, being the first cell type, with which circulating melanoma cells come into contact. Metastasis at the brain, therefore, requires disruption of and invasion through the brain endothelial cells. This study examines the barrier integrity of human brain endothelial cells upon addition of various human melanoma lines to investigate the mechanism of melanoma metastases at the brain (extravasation). Methods: In this study, Electric Cell-substrate Impedance Sensing (ECIS), amongst other impedance sensors, was used to assess changes in human brain endothelial barrier function upon the addition of various human melanoma lines. Imaging techniques such as immunocytochemistry and live cell imaging were used to visualise the melanoma interaction with the endothelium. Flow cytometry and proteomic profilers were used to characterize the melanoma lines for expression of proteins that may facilitate metastasis. Inhibition and co-culture assays were conducted to assess the involvement of several molecules expressed by the melanoma cells, in mediating brain endothelial barrier disruption. Results: Each melanoma line induced brain endothelial barrier disruption within the first few hours of addition. This disruption was observed in the paracellular component of the endothelial barrier before the basolateral component, suggesting a paracellular route of entry for melanoma invasion. Melanoma cells highly expressed various proteins commonly implicated in metastasis. However, these were not directly involved in mediating the barrier disruption seen with the melanoma cells. Intriguingly, a small subpopulation of melanoma cells upregulated the cell-surface molecule CD31, only after co-culture with the endothelial cells. Melanoma cells also produced a secondary cell type that disrupted the endothelial barrier considerably more drastically than their parent melanoma line. Discussion: Most studies consider the aberrant and constitutive expression of metastatic mediators in melanoma progression. Herein, there is evidence suggesting that melanoma metastasis may require just a few cells to adapt to the environment, phenotypically change and express specific proteins to initiate extravasation.